A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.
Bottom Line: We identified a transient interaction between our model substrates and Tom40.Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22.Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.
Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.Show MeSH
Mentions: We investigated whether TOM is engaged in a possible alternative route for MIA-dependent proteins. The core of the TOM complex is formed by the β-barrel protein Tom40, which forms a channel for protein import (Pfanner et al., 2004; Neupert and Herrmann, 2007; Schmidt et al., 2010; Endo et al., 2011; Dimmer and Rapaport, 2012; Qiu et al., 2013). Cysteine residues that are positioned such that they face the lumen of the channel should be amenable to chemical modifications, which would create spatial hindrance and clog the Tom40 channel. On the basis of a recent study (Qiu et al., 2013), we used two yeast strains that harbor Tom40 with the cysteine residues introduced in positions 89/360 and 130/138, in addition to the wild-type strain and a strain with Tom40 that lacked native cysteine residues (Tom40CFREE; Supplemental Figure S1A). The levels of mitochondrial proteins in the mutant strains were unaffected (Supplemental Figure S1B). We blocked the Tom40 channel by applying the alkylating agent methoxypolyethylene glycol maleimide (mPEG; molecular weight, 5 kDa). This compound reacts with accessible cysteine thiol groups. In intact mitochondria, mPEG modified cysteine residues of the Tom40 mutants (Figure 3A, lanes 4 and 8) but did not affect Tom40CFREE or wild-type Tom40 with native cysteine residues, indicating that native cysteine residues were not accessible for modification (Figure 3A, lanes 2 and 6). Given the presence of cysteine residues in the cytosolic domain, Tom70 was shifted due to mPEG modification, whereas Tom22 and Tom20 remained unmodified (Figure 3A). The mPEG modification of the Tom40 mutants with the thiol groups facing the channel resulted in a change in TOM complex migration in blue-native gel (Figure 3B, lanes 4 and 8). These experiments verified the specificity of mPEG treatment.
Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.